Translocation and metabolism of glycine betaine in nodulated alfalfa plants subjected to salt stress

The fate of radioactive glycine betaine was investigated in 31-day-old alfalfa ( Medicago sativa L. cv Europe) plants nodulated by Rhizobium meliloti 102 F 34. Radioactive [methyl-¹⁴C]- or [1,2-¹⁴C]glycine betaine was fed for 6 h to plants subjected or not to stress by 0.2 M NaCl. A 36% decrease in glycine betaine uptake was observed in salinized plants. No loss of radioactivity in the gas phase or the growth medium was ever observed from either stressed or unstressed plants, even after a 4-day chase period. Glycine betaine catabolism was negligible in shoots of both control and salinized plants, but it was important in roots and even more significant in nodules of unstressed plants. In unstressed nodules, 52% of the labelled betaine was metabolized after 4 days, and the half-life of glycine betaine was estimated at ca 4 days. On the contrary, catabolism was dramatically reduced in stressed roots and, particularly, nodules in which the half-life of glycine betaine increased to at least 16 days. Analysis of the redistribution of radioactivity among plant organs during the chase period shows that glycine betaine was translocated from the roots to the nodules of salinized plants, so that during this period salinization resulted in a 91% increase in nodule radioactivity, whereas a 34% decrease was observed in control plants. Altogether, reduced catabolism and increased translocation of glycine betaine to stressed nodules favored its accumulation in these organs. The high level of glycine betaine might contribute to maintain a better water status in the nodule and, thus, protect the nitrogen fixation activity against the deleterious effects of elevated osmolarity in the nutrient solution.     

Maize Rabl7 overexpression in Arabidopsis plants promotes osmotic stress tolerance

Rabl7 is a Late Embryogenesis Abundant (LEA) protein from maize, which accumulates largely during embryogenesis and also in vegetative tissues when subjected to stress conditions. We have analysed the effect of Rab 17 expression under a constitutive promoter in vegetative tissues of transgenic Arabidopsis thaliana plants. These transgenic plants have higher sugar and proline contents, and also higher water loss rate under water stress. In addition, these plants are more tolerant than non-transformed controls to high salinity and recover faster from mannitol treatment. Our results point to a protective effect of Rabl7 protein in vegetative tissues under osmotic stress conditions.     

CO2 Assimilation and Water Relations of Almond Tree (Prunus amygdalus Batsch) Cultivars Grown Under Field Conditions

Gas exchanges and leaf water potential (_w) of six-years-old trees of fourteen Prunus amygdalus cultivars, grafted on GF-677, were studied in May, when fruits were in active growing period, and in October, after harvesting. The trees were grown in the field under rain fed conditions. Predawn _w showed lower water availability in October compared with May. The lowest _w values at midday in May increased gradually afterwards, while in October they decreased progressively until night, suggesting a higher difficulty to compensate the water lost by transpiration. However, relative water content (RWC) measured in the morning was similar in both periods, most likely due to some rainfall that occurred in September and first days of October that could be enough to re-hydrate canopy without significantly increasing soil water availability. The highest net photosynthetic rate (P _N) was found in both periods early in the morning (08:00–11:00). Reductions in P _N from May to October occurred in most cultivars except in José Dias and Ferrastar. In all cultivars a decrease in stomatal conductance (g _s) was observed. Photosynthetic capacity (P _max) did not significantly change from spring to autumn in nine cultivars, revealing a high resistance of photosynthetic machinery of this species to environmental stresses, namely high temperature and drought. Osmotic adjustment was observed in some cultivars, which showed reductions of ca. 23 % (Duro d' Estrada, José Dias) and 15 % (Tuono) in leaf osmotic potential (). Such decreases were accompanied by soluble sugars accumulation. The Portuguese cultivar José Dias had a higher photosynthetic performance than the remaining genotypes.     

Proline accumulation and nitrate reductase activity in contrasting sorghum lines during mid-season drought stress

Six lines of sorghum ( Sorghum bicolor L. Moench) with differing drought resistance (IS 22380, ICSV 213, IS 13441 and SPH 263, resistant and IS 12739 and IS 12744, susceptible) were grown under field conditions in the semi-arid tropics and analysed for proline and nitrate reductase activity (NRA; EC 1.6.6.1) during a mid-season drought. The resistant lines accumulated high levels of proline, while the susceptible lines showed no significant proline accumulation. Most of the proline was accumulated after growth of the plants had ceased. In a separate greenhouse experiment, most of the proline was found in the green rather than the fired portions of leaves. The levels returned to that of irrigated controls within 5 days of rewatering. Proline levels increased as leaf water potential and relative water content fell, and there was no apparent difference among the different sorghum lines with change in plant water status. Susceptible lines accumulated less proline than resistant lines as leaf death occurred at higher water potentials. Proline accumulation may, however, contribute to the immediate recovery of plants from drought. Leaf NRA reached high levels at about 35 days after sowing in both the stressed and irrigated plants, after which it declined. The decline in NRA was more pronounced in the stressed than in the irrigated plants and closely followed changes in the growth rate. Upon rewatering, NRA increased several-fold in all the lines and, in contrast to proline accumulation, genotypic differences in NRA were small, both during stress and upon rewatering. The high sensitivity of NRA to mild drought stress was reflected in the rapid decline of activity with small changes in leaf water potential and relative water content. The results are discussed in the light of a possible role for proline during recovery from drought, and the maintenance of NRA during stress and its recovery upon rewatering.     

Role of proline and leaf expansion rate in the recovery of stressed white clover leaves with increased phosphorus concentration

Osmotic adjustment (OA) and increased cell-wall extensibility required for expansive leaf growth are well defined components of adaptation to water stress in dry soil, which might interact with soil phosphorus (P) concentration and defoliation frequency for intensively grazed white clover in legume-based pastures. Experiments were conducted with frequently and infrequently defoliated mini-swards of white clover growing in dry soil with low and high P concentrations. The higher yielding high-P plants were able to dry the soil to greater soil water suctions; their leaves had lower water potential values, yet they showed fewer water stress symptoms and underwent a more complete recovery from the water stress symptoms on rewatering, than the low-P plants. High- P plants had greater OA, proline concentration and leaf expansion rate. On the other hand, low-P plants showed an increased osmotic concentration when there was no change in the total solute content per unit of leaf d. wt, indicating more loss of water from the leaf tissue. The key measures that appeared to be directly associated with plant recovery over a short period following water stress were increased proline concentration and leaf expansion rate, probably resulting from increased cell-wall extensibility rather than increased production of cells for the high-P plants.     

Effects of salt stress on growth, mineral nutrition and proline accumulation in relation to osmotic adjustment in rice (Oryza sativa L.) cultivars differing in salinity resistance

Salt-resistant rice cultivars Nona Bokra and IR 4630 exposed at the seedling stage during one or two weeks to 0, 20, 30, 40 or 50 mM NaCl accumulated less Na, Cl, Zn and proline and more K at root and shoot levels than salt-sensitive I Kong Pao and IR 31785. Aiwu, a moderately resistant genotype, exhibited an intermediate behaviour. P transport from root to shoot was inhibited in the most sensitive cultivar IR 31785. Accumulation of Na and Cl and decrease in K content at the shoot level were restricted to the oldest leaves in salt-resistant genotypes while proline accumulated in the youngest leaves in all cultivars. In the presence of NaCl, the osmotic potentials of the roots and of the oldest and youngest leaves were lower in the salt-resistant than in the salt-sensitive genotypes, differences among genotypes increasing with stress intensity. Proline did not appear to be involved in osmotic adjustment in salt-stressed rice plants and the significance of its accumulation is discussed in relation to salinity resistance.Abbreviations cv(s). cultivar(s) - EC electrical conductivity - IKP I Kong Pao - J rate of ion transport - MCW methanol-chloroform-water - PAR photon flux density - Pc partitioning coefficient - RGR mean relative growth rate - RI resistance index - s osmotic potential     

Phosphoenolpynivate carboxylase, malate and alcohol dehydrogenase activities in alfalfa (Medicago sativa) nodules under water stress

The effect of drought upon phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31), malate ddiydrogenase (MDH; EC 1.1.1.37), alcohol dehydrogenase (ADH; EC 1.1.1.1) and β -hydroxybulyrate dehydrogenase ( β -OH-BDH; EC 1.1.1.30) enzyme activities as well as the leghemoglobin (Lb), malate and ethanol contents of alfalfa nodules ( Medicago sativa L. cv. Aragon) were examined. Both the ieghemoglobin (Lb) content and the Lb/soluble protein ratio were significantly reduced at a nodule water potential (Ψ_nod) of—1.3 MPa. At lower Ψ_nod, Lb content decreased further, but the ratio remained unchanged. Slight stress (—1.3 MPa) drastically affected acetylene reduction activity (ARA; 60% reduction) whereas in vitro PEPC activity was main-tained at relatively constant values. As stress progressed (—2.0 MPa), a simultaneous reduction in both activities was observed. Severe stress (Ψ_nod lower than —2.0 MPa) stimulated in vitro PEPC. Bacteroid β -J-OH-BDH activity was stimulated by slight (—1.3 MPa) and moderate (—2.0 MPa) drought. MDH activity rose in slightly stressed nodules (Ψ_nod—1.3 MPa). Greater water deficits sharply decreased MDH activity to values significantly lower than those found in control nodules. Nodule malate content followed the same pattern as MDH. The plant fraction of the nodule showed constitutive ADH activity and contained ethanol. ADH was stimulated at slight (— 1.3 MPa) and moderate drought levels (—2.0 MPa). Ethanol content showed similar behavior to ADH activity. Inhibition of ARA, reduction of Lb content and stimulation of the fermentative metabolism induced by water stress suggest some reduction ira O₂ availability within the nodule.     

In vitro-selection of hydroxyproline-resistant cell lines of wheat (Triticum aestivum): accumulation of proline, decrease in osmotic potential, and increase in frost tolerance

Five hundred hydroxyproline-resistant cell lines were selected from cell cultures of wheat ( Triticum aestivum L. cv. Koga II) after plating on 10 to 30 m M hydroxyproline (Hyp) containing solid Gamborg B 5 medium. All selected cell lines from 30 m M Hyp-medium contained increased (up to 17-fold) levels of free proline. Seventy-four cell lines were transferred to Hyp-free medium and subcultivated 25 times, for 12 months altogether, after which 80% still had increased proline levels. Fourteen cell lines with increased proline levels were further investigated in liquid media with regard to their frost tolerance, which was measured by means of electrolyte leakage. Ten of them showed increased fros tolerance, with LT 50 values as low as 2.7°C below that of the wild type (-4.7°C). Besides increased proline levels and increased percentage dry weight, the Hyp-resistant cell lines had lower osmotic potentials. Osmotic potentials correlated better than levels of free proline with the increase in frost tolerance.     

Growth, Water Content, and Proline Accumulation in Drought-Stressed Callus of Date Palm

This study was conducted to examine the response of date palm (Phoenix dactylifera L., cvs. Barhee and Hillali) calli to water stress. Callus derived from shoot tip explants was inoculated in liquid Murashige and Skoog medium containing 10 mg dm^–3-naphthaleneacetic acid, 1.5 mg dm^–3 2-isopentenyladenine, and 0 to 30 % (m/v) polyethylene glycol (PEG 8000) to examine the effect of water stress. After 2 weeks, callus growth, water content, and proline accumulation were assessed. Increasing water stress caused a progressive reduction in growth as expressed in callus fresh mass, relative growth rate, and index of tolerance. Both genotypes tested followed this general trend, however, cv. Barhee was more tolerant to drought stress than cv. Hillali. Increasing PEG concentration was also associated with a progressive reduction in water content and increased content of endogenous free proline.     

Osmotic adjustment inSorghum bicolor (L. Moench) grown under moisture stress in soil and osmotically modified solution cultures

Sorghum (Sorghum bicolor L. Moench) plants were grown in solution culture and stressed at three rates of decreasing leaf water potential (−0.123, −0.068 and −0.029 MPa day⁻¹) achieved by the incremental addition of an osmoticum, polyethylene glycol (PEG) 6000 to the solutions. Plants were also grown in soil and given different amounts of water which resulted in rates of decreasing leaf water potentials of −0.130 and −0.073 MPa day⁻¹. The rate of stress and the culture system influenced the accumulation of solutes in the cell, but not cell volume. A rapid stress (−0.123 and −0.130 MPa day⁻¹) to approximately −1.6 MPa leaf water potential resulted in 0.75 and 0.16 MPa of osmotic adjustment in the PEG and soil culture respectively. At moderate stress (−0.068 and −0.073 MPa day⁻¹) respective values were 1.68 and 0.58 MPa. There were some visual symptoms in the solution grown plants characteristic of uptake of high molecular weight PEG. However the relative growth rates of these plants were equal to or greater than those of the soil grown plants. In view of the differences in plant water status of soil and PEG solution cultured plants it was concluded that the use of the latter system would not be entirely suitable for some studies of drought resistance in sorghum, as related to crop performance in the field.     

An inland and a coastal population of the Mediterranean xero-halophyte species Atriplex halimus L. differ in their ability to accumulate proline and glycinebetaine in response to salinity and water stress

Soil salinity and drought compromise water uptake and lead toosmotic adjustment in xero-halophyte plant species. These importantenvironmental constraints may also have specific effects onplant physiology. Stress-induced accumulation of osmocompatiblesolutes was analysed in two Tunisian populations of the Mediteraneanshrub Atriplex halimus L.—plants originating from a salt-affectedcoastal site (Monastir) or from a non-saline semi-arid area(Sbikha)—were exposed to nutrient solution containingeither low (40 mM) or high (160 mM) doses of NaCl or 15% polyethyleneglycol. The low NaCl dose stimulated plant growth in both populations.Plants from Monastir were more resistant to high salinity andexhibited a greater ability to produce glycinebetaine in responseto salt stress. Conversely, plants from Sbikha were more resistantto water stress and displayed a higher rate of proline accumulation.Proline accumulated as early as 24 h after stress impositionand such accumulation was reversible. By contrast, glycinebetaineconcentration culminated after 10 d of stress and did not decreaseafter the stress relief. The highest salt resistance of Monastirplants was not due to a lower rate of Na⁺ absorption; plantsfrom this population exhibited a higher stomatal conductanceand a prodigal water-use strategy leading to lower water-useefficiency than plants from Sbikha. Exogenous application ofproline (1 mM) improved the level of drought resistance in Monastirplants through a decrease in oxidative stress quantified bythe malondialdehyde concentration, while the exogenous applicationof glycinebetaine improved the salinity resistance of Sbikhaplants through a positive effect on photosystem II efficiency. Key words: Atriplex halimus, glycinebetaine, halophyte, NaCl, osmotic adjustment, proline, salinity, water stress     

Drought tolerance in faster- and slower-growing black spruce (Picea mariana) progenies: II. Osmotic adjustment and changes of soluble carbohydrates and amino acids under osmotic stress

The possibility was considered that osmotic adjustment, the ability to accumulate solutes in response to water stress, may contribute to growth rate differences among closely-related genotypes of trees. Progeny variation in osmotic adjustment and turgor regulation was investigated by comparing changes in osmotic and pressure potentials, soluble carbohydrates, and amino acids in osmotically stressed seedlings in 4 full-sib progenies of black spruce [ Picea mariana (Mill.) B. S. P.] that differed in growth rate under drought. Osmotic stress was induced by a stepwise increase in the concentration of polyethylene glycol (PEG)-3350 from 10 (w/v) to 18 and 25%, which provided osmotic potentials in solution culture of -0.4, -1.0 and -2.0 MPa each for 3 days. All 4 progenies maintained a positive cell turgor even at 25% PEG, due to a significant decline in osmotic potential. Although total amino acids, principally proline, increased, ca 60% of the decrease in osmotic potential was attributable to soluble carbohydrates and glucose was the major osmoregulating solute. There was little progeny variation in any of measured parameters in unstressed seedlings. Compared to two slower-growing progenies, the two progenies capable of more vigorous growth under drought in the field accumulated more soluble carbohydrates (mainly glucose and fructose), developed lower osmotic potential and maintained higher turgor pressure when osmotically-stressed in solution culture. The ability to adjust osmotically and maintain turgor under drought stress could thus be a useful criterion for the early selection of faster-growing, drought-tolerant genotypes.     

Water stress induced alterations in ornithine aminotransferase of ragi (Eleusine coracana): Protection by proline against heat inactivation and denaturation by urea and guanidinium chloride

Water stress resulted in a specific response leading to a large and significant increase (80-fold) in free proline content of ragi (Eleusine coracana leaves and seedlings. L-Proline protected ornithine aminotransferase, an enzyme in the pathway for proline biosynthesis, isolated from normal and stressed ragi leaves against heat inactivation and denaturation by urea and guanidinium chloride. The protection of the stressed enzyme by L-proline was much more complete than that of the enzyme isolated from normal leaves. While L-ornithine, one of the substrates, protected the stressed enzyme against inactivation, it enhanced the rate of inactivation of the normal enzyme. α-Ketoglutarate protected both the normal and stressed enzyme against inactivation and denaturation. These results support the suggestion that ornithine aminotransferase has undergone a structural alteration during water stress. In view of the causal relationship between elevated temperature and water stress of plants under natural conditions, the protection afforded by proline against inactivation and denaturation of the enzyme from stressed leaves assumes significance. These results provide an explanation for a possible functional importance of proline accumulation during water stress.     

Osmotic adjustment of chickpea (Cicer arietinum) is not associated with changes in carbohydrate composition or leaf gas exchange under drought

Genetic differences in osmotic adjustment (OA) have been reported among chickpea (Cicer arietinum) cultivars. In this study eight advanced breeding lines (ABLs) derived from a cross between CTS 60543 (high OA) and Kaniva (low OA) and Tyson (medium OA) and Kaniva, along with the parents, were evaluated for OA, leaf carbohydrate composition and leaf gas exchange under dryland field conditions in India. The water potential (WP) decreased to lower values (less than −2.5 MPa) in Tyson, M 110 and M 86 than in the other genotypes. With decrease in WP, OA increased by 0.5 MPa in Kaniva and CTS 60543 to 1.3 MPa in M 55. As the decrease in WP varied with genotype, when OA was regressed against WP M 39 and M 55 had greater increases in OA with decrease in WP than the remaining nine genotypes, including the parents. As WP decreased, leaf starch content decreased while total soluble sugars, hexoses and sucrose increased: the decrease in starch was much smaller in M 93 and M 129 than in Tyson and M 51, but genotypic differences could not be detected in the increase in total sugars, hexoses or sucrose. The rates of photosynthesis and transpiration decreased as the WP became more negative, but M 129 reached low rates of photosynthesis (2 μmol m⁻² s⁻¹) and transpiration at a WP of −1.7 MPa, whereas Tyson reached the same low rate at −2.4 MPa. While OA varied among the chickpea genotypes, the differences were not associated with the changes in carbohydrate composition or the rates of gas exchange at low values of WP. Further, the degree of OA of the 11 genotypes was not the same as when they were selected for differences in OA under rainout shelter conditions in the field in Australia, suggesting that OA may show poor stability depending upon the stress level, location or physiological stage of the plant. This suggests that OA is not a valuable drought-resistance trait to select for in chickpea breeding programmes.     

Influence of Phosphorus Application on Water Relations, Biochemical Parameters and Gum Content in Cluster Bean Under Water Deficit

Relative water content (RWC), leaf water potential (_w) and osmotic potential (_s), contents of chlorophyll (Chl) a, Chl b, soluble sugars, and seed quality (gum content) were used to evaluate the role of phosphorus in alleviation of the deleterious effect of water deficit in clusterbean (Cyamopsis tetragonoloba L. Taub). Under water stress, _w, _s, and Chl and gum contents decreased and soluble sugar contents increased. Phosphorus application increased Chl and sugar contents in control plants and ameliorated negative effects of water stress.     

Water relations and leaf gas exchange properties in some elite canola (Brassica napus) lines under salt stress

In order to examine whether growth of eight genetically diverse canola (Brassica napus) lines under salt stress is positively associated with their rate of photosynthesis and other gas exchange related attributes, 20‐day old plants of all eight lines were subjected to salinised soil containing 2.4 dS m^?1 NaCl (control), 4 dS m^?1 NaCl, 8 dS m^?1 NaCl or 12 dS m‐¹ NaCl. The lines DGL (non canola) and Dunkeld were found to be salt tolerant and Rainbow and Cyclon salt sensitive with regard to shoot dry matter production and seed yield under saline conditions. In most of the lines there was a negative relationship between growth and net CO₂ assimilation rate. For example, the salt sensitive line Cyclon was the lowest and Con‐III the highest, and the salt tolerant line Dunkeld intermediate in net CO₂ assimilation rate under salt stress. Stomatal conductance was found to be lower in the salt sensitive line Cyclon, followed by the salt tolerant line Dunkeld along with Oscar. Water use efficiency estimated as Pn/E was moderate in the salt sensitive line Cyclon and the salt tolerant line Dunkeld. In conclusion, high salt tolerance of Dunkeld and DGL (non‐canola) was not positively associated with net CO₂ assimilation rate or Pn/E.     

The pattern of distribution of pectin, peroxidase and lignin in the middle lamella of secondary xylem fibres in alfalfa (Medicago sativa)

BACKGROUNDS AND AIMS: Information on the micro-distribution of lignin within the middle lamella is only just beginning to emerge. This paper provides evidence of marked heterogeneity in the micro-distribution of lignin, pectin, peroxidase and hydrogen peroxide in the middle lamella of alfalfa (Medicago sativa). METHODS: Specimens from alfalfa stems were collected and processed for transmission electron microscopy. The middle lamella architecture was examined prior to and during lignification, using transmission electron microscopy in combination with pectin- and lignin-specific staining. In addition, immuno-gold labelling of peroxidase and cytochemical localization of hydrogen peroxide (H2O2) were undertaken. KEY RESULTS: Lignin showed inhomogeneity in its distribution in the middle lamella. It was found that the distribution of pectin was irregular and corresponded to the pattern of deposited lignin. Additionally, a similarity in the pattern of the deposited lignin to the pattern of distribution of peroxidase and H2O2 was also observed. CONCLUSIONS: Irregular distribution of pectin in the middle lamella may be related to subsequent inhomegeneity in lignin in this region.